Tran, Therese

Abstract [en]

Kromasil is a silica-based packing material used in analytical and preparative HPLC columns for analysis and purification of pharmaceutical substances. Derivatised silica intended for use with polar mobile phases is known as Reversed Phase silica (RP-silica). This work is based on Wide Pore (300Å) RP-silica C4. Larger pores and shorter alkylchain lengths give the possibility to separate larger biomolecules, such as peptides and proteins,efficiently.
This paper has a number of purposes. The primary purpose was to develop and optimise packing methods for two particles size of Kromasil 300, 13 and 25µm. The second purpose included examining the mechanical and chemical stability of Kromasil and determination of the pore availability and the geometry of the pore system. A comparison of Kromasil with other materials from competing companies Vydac (10-15µm), Jupiter (13µm), YMC (15µm), Daiso (15µm) and Polymer Labs (10-15µm) is also presented.
Materials used in optimising packing methods are toluene as analyte and ACN/H2O 70:30 as the mobile phase. The following results were obtained:
13µm >= 30 000 plates per meter at a flow of 0.50ml/ min
25µm ª 20 000 plates per meter at a flow of 0.30 ml/min
The reproducibility was acceptable. Furthermore, tests with Kromasil indicated good chromatographic properties.
Concerning mechanical stability, Kromasil, YMC and Daiso were comparable while Jupiter and Vydac were inferior. The least mechanically stable material was Polymer Labs, a polymer-based material. This might be due to the fact that the testing method is more suitable for silica-based materials and not polymer-based materials.
The chemical stability was tested by measuring silica and silane leakage from the materials exposed to different eluent pH (acid, neutral and alkaline). The leakage is due to the process of hydrolysis. Results from AAS (Atomic Absorption Spectrometry) showed that most of the materials had silica leakage under 100 ppm, and indication of excellent chemical stability. Vydac differed significantly from the other materials and had the highest silica and silane leakage, thereby the least chemically stable material. Since Polymer Labs is polymer-based, the material was not affected by hydrolysis.
SEC (size Exclusion Chromatography) was used in determining pore-availability and the geometry of the pore system. Kromasil and Daiso were the best materials in terms of mass transport. Jupiter, YMC and Vydac appeared to have good pore availability, however they were dependent on the flow. This indicated problem with the mass transport. Polymer Labs had the highest excluded molecular weight at a low flow, but at a higher flow the result was not comparable.